Agrobacterium Tumefaciens As an Agent of Disease

380 Review TRENDS in Plant Science Vol.8 No.8 August 2003 Agrobacterium tumefaciens as an agent of disease Matthew A. Escobar1 and Abhaya M. Dandekar2 1Department of Cell and Organism Biology, Lund University, So¨ lvegatan 35, Lund, SE-22362, Sweden 2Department of Pomology, University of California, 1 Shields Ave, Davis, CA 95616, USA Twenty-six years ago it was found that the common lesions on grape roots [4]. A. vitis can survive in planta in soil bacterium Agrobacterium tumefaciens is capable of the intercellular spaces of grape tissue without causing extraordinary feats of interkingdom genetic transfer. disease but will initiate tumorigenesis upon tissue Since this discovery, A. tumefaciens has served as a wounding (most commonly frost injury) [4]. Erwin Smith model system for the study of type IV bacterial and C.O. Townsend [5] reported 10 years after the secretory systems, horizontal gene transfer and bac- discovery of A. vitis that Bacterium tumefaciens (now terial–plant signal exchange. It has also been modified Agrobacterium tumefaciens) was the causal agent of crown for controlled genetic transformation of plants, a core gall disease in Paris daisy. This organism is capable of technology of plant molecular biology. These areas inducing tumors at wound sites on the stems, crowns and have often overshadowed its role as a serious, wide- roots of hundreds of dicots, although root necrosis is not spread phytopathogen – the primary driver of the first characteristic of A. tumefaciens-mediated crown gall 80 years of Agrobacterium research. Now, the diverse disease [6]. The pioneering work of Cavara and Smith areas of A. tumefaciens research are again converging and Townsend ushered in a century of study of Agrobac- because new discoveries in transformation biology and terium as an agent of disease, a model system of horizontal the use of A. tumefaciens vectors are allowing the gene transfer and a tool for plant transformation (Table 1). development of novel, effective biotechnology-based Although crown gall disease is not generally fatal strategies for the control of crown gall disease. unless infection occurs in young plants, crown gall related reduction in crop yield and/or vigor can be significant in Members of the genus Agrobacterium are ubiquitous many perennial horticultural crops [7], such as grape [8], components of the soil microflora, the vast majority of apple [9] and cherry [10]. The decreased productivity of which are saprophytic, surviving primarily on decaying galled plants is probably caused by several factors, organic matter. However, several species of agrobacteria including decreased water and nutrient flow owing to cause neoplastic diseases in plants, including Agrobacter- damaged or constricted vasculature at the site of gall ium rhizogenes (hairy root disease), Agrobacterium rubi development, and significant water and nutrient allo- (cane gall disease), Agrobacterium tumefaciens (crown gall cation to the rapidly dividing but unproductive gall sink disease) and Agrobacterium vitis (crown gall of grape). [11–13]. In addition, crown galls are sites for secondary During the past 26 years, it has become apparent that infection by other phytopathogens (e.g. Pseudomonas Agrobacterium pathogenesis is a unique and highly syringae and Armillarea mellea) or pests (insect borers), specialized process involving bacterium–plant interking- and can increase plant susceptibility to abiotic stresses dom gene transfer [1]. Crown gall and hairy root have been [6,8,12]. Finally, in planta populations of tumorigenic described as a form of ‘genetic colonization’ [2] in which the agrobacteria can negatively affect graft take, because transfer and expression of a suite of Agrobacterium genes tumor tissue developing at the graft union prevents fusion in a plant cell causes uncontrolled cell proliferation and of stock and scion tissues [8]. the synthesis of nutritive compounds that can be metab- olized specifically by the infecting bacteria. Thus, infection Disease process – transformation and tumorigenesis effectively creates a new niche specifically suited to Agrobacterium pathogenesis requires two basic elements: Agrobacterium survival. This article focuses specifically (1) delivery of tumorigenic DNA into the plant genome on crown gall, the most agriculturally significant disease (transformation); and (2) the resultant alteration of plant caused by agrobacteria, and the state of current and future cell metabolism, resulting in cell proliferation and the strategies for crown gall disease control. synthesis of nutritive compounds that provide a selective Fridiano Cavara first identified a flagellate, bacilloid advantage for Agrobacterium (tumorigenesis). The focus bacterium (termed Bacillus ampelopsorae) as the causal hereisentirelyonthegall-formingagrobacteria;seeRef.[14] agent of crown gall of grape in 1897 [3]. This organism, for a review of A. rhizogenes and hairy root disease. now called A. vitis, causes the growth of neoplastic tumors on the stem and crown of grapevines and induces necrotic From chemotaxis to integration A detailed treatment of Agrobacterium transformation is Corresponding author: Abhaya M. Dandekar ([email protected]). beyond the scope of this article but several reviews from http://plants.trends.com 1360-1385/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00162-6 Review TRENDS in Plant Science Vol.8 No.8 August 2003 381 Table 1. Selected discoveries and insights in Agrobacterium biology Year Discovery Refs 1853 First written report of crown gall disease. [72] 1897 Agrobacterium vitis identified as the causal agent of crown gall in grape. [3] 1907 Agrobacterium tumefaciens identified as causal agent of crown gall in Paris daisy (Argyranthemum frutescens). [5] 1947 Sterile plant tumor tissue can proliferate indefinitely on hormone-free medium in culture. Tumor cells are [38] proposed to be ‘transformed’ by an Agrobacterium-derived tumor-inducing principle (TIP). 1956 Unusual low molecular weight nitrogenous compounds (opines) are identified exclusively in tumor tissue. [73] 1971 Agrobacterium tumefaciens loses virulence when grown at 378C. The TIP can be transferred between virulent [74,75] and avirulent A. tumefaciens strains. 1974 Agrobacterium tumefaciens virulence depends on the presence of a large ‘tumor-inducing’ (Ti) pasmid. The TIP [76] is probably a component of the Ti plasmid. 1977 The T-DNA region of the Ti plasmid is present in the genome of crown gall tumor cells: the T-DNA is the TIP. [1] 1980 The opine concept: the synthesis of opines by transformed cells creates an ecological niche for the infecting [34] strain of Agrobacterium. 1983 First plant transformed with a recombinant gene using Agrobacterium tumefaciens as a vector. [77] 1984 T-DNA oncogenes are identified that mediate overproduction of auxin and cytokinin. [26,27] 1985 The virA/virG two-component regulatory system is identified as a central component of signal perception and [20] transduction in Agrobacterium transformation. 1986– Further elucidation of the vir-gene-encoded T-DNA transfer process; identification of plant genes involved in [16–18,47,78,79] present Agrobacterium tumefaciens transformation; extension of A. tumefaciens host range for transformation of monocots; sequencing of the A. tumefaciens (C58) genome. both bacterial [15,16] and plant [17,18] perspectives have genomethroughnon-homologousrecombinationinaprocess recently been published. Briefly, amino acids, organic acids that appears to require plant-encoded proteins (probably and sugars released from wounded plant cells act as enzymes related to DNA repair or recombination) [22,23]. chemoattractants to tumorigenic agrobacteria, which bind to plant cells in a polar orientation upon reaching the Forming a gall in planta wound site [17,19]. Weak attachment to the plant cell is Genes present in the Agrobacterium T-DNA possess the cis first achieved through synthesis of acetylated polysacchar- motifs (e.g. TATA box, CAAT box, polyadenylation signal) ides, followed by strong binding through the extrusion of required for expression in the eukaryotic plant host [24]. cellulose fibrils [17]. Simultaneously, the vir regulon, a set There are two general classes of genes in T-DNA: of operons required for the transfer of virulent DNA, is oncogenes and opine-related genes. The oncogenes alter activated by the VirA/VirG two-component regulatory phytohormone synthesis and sensitivity in the infected system [20]. The presence of acidic extracellular conditions cell, thus generating the tumor phenotype. T-DNAs from (pH 5.0–5.5), phenolic compounds and monosaccharides octopine-type A. tumefaciens strains (e.g. 15955 and Ach5) at a plant wound site directly or indirectly induce contain five oncogenes: iaaM, iaaH, ipt, 6b and 5 [25]. The autophosphorylation of the transmembrane receptor tryptophan mono-oxygenase iaaM converts tryptophan kinase VirA [19]. Activated VirA transfers its phosphate into indole-3-acetamide, and the indoleacetamide hydro- to the cytoplasmic VirG protein, which then binds to the vir lase iaaH catalyzes the synthesis of the plant hormone box enhancer elements in the promoters of the virA, virB, indole acetic acid (IAA) from indole-3-acetamide (Fig. 1) virC, virD, virE and virG operons, upregulating transcrip- [26]. The ipt gene product mediates the condensation of tion [15]. Through the co-operative action of the VirD1 and adenosine monophosphate with isopentenyl pyrophos- VirD2 proteins, a single-stranded DNA fragment (the

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